1. Technical Field
The field of this invention is cDNA libraries.
2. Background of the Invention
A complementary DNA or cDNA is a deoxyribonucleic acid that contains the information coding for the synthesis of proteins, but lacks the intervening introns present in genomic DNA. The synthesis of cDNA, the use of cDNA and libraries of cDNA play a critical role in a variety of different application in biotechnology and related fields. Applications in which cDNAs and/or libraries thereof are employed include gene discovery, differential gene expression analysis, and the like. A variety of protocols have been developed to prepare cDNA and libraries thereof, where such methods are continually being modified.
In standard methods currently used for the preparation of cDNA libraries, the mRNA in the cell is isolated by virtue of the presence of a polyadenylated tail present at its 3xe2x80x2 end which binds to a resin specific for this structure (oligo dT-chromatography). The purified mRNA is then copied into cDNA using a reverse transcriptase, which starts at the 3xe2x80x2 end of the mRNA and proceeds towards the 5xe2x80x2 end. Second strand synthesis is then performed. Linkers are added to the ends of the double stranded cDNA to allow for its packaging into virus or cloning into plasmids. At this stage, the cDNA is in a form that can be propagated.
One disadvantage observed with current cDNA library synthesis protocols is that current methods tend to produce libraries having a significant proportion of incomplete cDNAs, which results from inefficiencies in the reverse transcriptase employed to generate the library. To compensate for the incomplete cDNA constituents of the library, investigators must perform many rounds of isolation (screenings) and construct a xe2x80x9cfull-lengthxe2x80x9d cDNA from the accumulated pieces. Such processes are resource intensive and do not ensure that each initial mRNA is represented in the cDNA library.
In addition, there is significant under-representation of sequences close to the 5xe2x80x2 end of mRNAs since in cDNA libraries produced by convention methods. This under-representation results from the fact that the reverse transcriptase will usually xe2x80x9cfall offxe2x80x9d before reaching these sequences.
Another problem concerning cDNA synthesis is the source and quality of the mRNA used. Using present day technology, the mRNA that is used as a source for cDNA synthesis is purified by its 3xe2x80x2 end polyadenylated tail. However, some mRNAs do not possess a 3xe2x80x2 end but all mRNAs have a 5xe2x80x2 cap structure. Consequently, a cDNA library constructed from this source of mRNA would be more representative of the total genetic information present in the cell. In recent years, unsuccessful attempts have been made to develop antibodies directed against the cap structure of mRNA. The problems usually encountered were related to the insufficient affinity of the antibodies for the cap. This major drawback made it impossible to develop isolation protocols for capped mRNAs.
Therefore, there is continued interest in the development of new methods of cDNA synthesis.
Relevant Literature
U.S. patents of interest include U.S. Pat. No. 5,219,989.
Strategies for producing full length cDNAs are described in: Edery, et al., xe2x80x9cAn efficient strategy to isolate full-length cDNAs based on an mRNA cap retention procedure (CAPture),xe2x80x9d Mol Cell Biol (June, 1995)15(6):3363-71; Suzuki et al., xe2x80x9cConstruction and characterization of a full length-enriched and a 5xe2x80x2-end-enriched cDNA library,xe2x80x9d Gene (Oct. 24, 1997) 200(1-2):149-56; Alphey, xe2x80x9cPCR-based method for isolation of full-length clones and splice variants from cDNA libraries,xe2x80x9d Biotechniques (March 1997)22(3):481-4, 486; Carninci et al., xe2x80x9cHigh efficiency selection of full-length cDNA by improved biotinylated cap trapper,xe2x80x9d DNA Res (Feb. 28, 1997) 4(1):61-6; Carninci et al., xe2x80x9cHigh-efficiency full-length cDNA cloning by biotinylated CAP trapper,xe2x80x9d Genomics (Nov. 1, 1996)37(3):327-36; Schmid et al., xe2x80x9cA procedure for selective full length cDNA cloning of specific RNA species,xe2x80x9d Nucleic Acids Res (May 26, 1987)15(10):3987-96; Seki et al., xe2x80x9cHigh-efficiency cloning of Arabidopsis full-length cDNA by biotinylated CAP trapper,xe2x80x9d Plant J (September 1998) 15(5):707-20; Okayama et al., xe2x80x9cHigh-efficiency cloning of full-length cDNA,xe2x80x9d Mol Cell Biol (February 1982) 2(2):161-70; Sekine et al., xe2x80x9cSynthesis of full-length cDNA using DNA-capped mRNA,xe2x80x9d Nucleic Acids Symp Ser (1993) (29):143-4.
eIF-4E is described in Altmann et al., xe2x80x9cmRNA cap-binding protein: cloning of the gene encoding protein synthesis initiation factor eIF-4E from Saccharomyces cerevisiae,xe2x80x9d Mol Cell Biol (March 1987) 7(3):998-1003. eIF-4G is described in Hentze, Science (Jan. 24, 1997) 275: 500 and Haghighat et al., J. Biol. Chem. (Aug. 29, 1997) 272:21677.
Methods and compositions are provided for producing full-length cDNA libraries. In the subject methods, full length first strand cDNAs are isolated using a fusion protein of an eIF-4E domain and an eIF-4G domain separated by a flexible linker. Also provided is the novel fusion protein employed in the subject methods, as well as nucleic acids encoding, and host cells capable of expressing, the same. Finally, kits for use in practicing the subject methods are provided. The subject invention finds use in a variety of applications in which full-length cDNA libraries are employed.